There are only two types of molecules that can recognize antigens in a specific manner. One is immunoglobulin or antibody and the other is T cell receptor (TCR), which is α/β or γ/δ heterodimeric glycoprotein on cell membrane. The physical repertoire of TCR of immune system is generated in thymus through V (D)J recombination, followed by positive and negative selections. In peripheral environment, TCRs mediate the recognition of specific Major Histocompatibility Complex-peptide complexes (pMHC) by T cells and, as such, are essential to the immunological functioning of cells in the immune system.
TCR is the only receptor for presenting particular peptide antigens in Major Histocompatibility Complex (MHC). The exogenous or endogenous peptides may be the only sign of abnormality in a cell. In the immune system, once antigen-specific TCRs bind with pMHC complexes, it causes direct physical contact of a T-cell and an antigen presenting cell (APC). Then, the interaction of other membrane molecules in T cell and APC occurs and the subsequent cell signaling and other physiological responses are initiated so that a range of different antigen-specific T cells exert immune effects on their targets.
On T cell membrane, the TCR is associated with invariant proteins of CD3 involved in mediating signal transduction to form a complex. TCRs exist in many forms, which are structurally similar but T cells expressing them have quite distinct anatomical locations and probably have different functions. The extracellular portion of TCR consists of two membrane-proximal constant domains, and two membrane-distal variable domains. The variable domains contain polymorphic loops analogous to the complementary determining regions (CDRs) of antibodies. It is these loops that form the binding site of the TCR molecule and determine peptide specificity. The MHC class I and class II ligands corresponding to TCR are also immunoglobulin superfamily proteins but are specialized for antigen presentation, with a polymorphic peptide binding site which enables them to present a diverse array of short peptide fragments at the APC cell surface.
Like immunoglobulin (antibody) as a kind of antigen recognition molecule, TCR can be developed for diagnostic and therapeutic applications. However, it is difficult to produce proteins in (water) soluble form which are made up of more than one polypeptide subunit and have a transmembrane domain because, in many cases, the proteins are stabilized by their transmembrane region. This is the case for TCR, and is reflected in scientific literatures. It is reported that a truncated form of TCR containing either only extracellular domains or only extracellular and cytoplasmic domains can be recognized by TCR-specific antibodies, thus indicating that a partial region of recombinant TCR recognized by antibodies is correctly folded. However, the production is not high, and it is not stable and/or cannot recognize MHC-peptide complexes at a low concentration.
A Soluble TCR is useful, not only for research of TCR-pMHC interactions, but also potentially as a diagnostic tool to detect infection or as a marker for autoimmune diseases. Similarly, soluble TCRs can be used to deliver a therapeutic agent, e.g., a cytotoxic compound or an immunostimulating compound, to cells presenting a particular antigen, or to inhibit T cells, e.g., those reacting with an autoimmune peptide antigen. For these purposes, modification of TCR protein is important. Especially, it is very important for heterogeneous expression of TCRs in prokaryote or eukaryote systems.
As for expression of soluble TCR in E. coli, when TCR is separated from the membrane, instability and low protein yield are major hurdles for developing therapeutic or diagnostic reagents with TCR or its fragment. In order to overcome inherent instability of single-chain TCRs, production of a TCR heterodimer is described in some literatures, which includes a native disulfide bridge linking the respective subunits (Garboczi, et al., (1996), Nature 384 (6605): 134-41; Garboczi, et al., (1996), J Immunol 157(12): 5403-10; Chang et al., (1994), PNAS USA 91: 11408-11412; Davodeau et al., (1993), J. Biol. Chem. 268(21): 15455-15460; Golden et al., (1997), J. Imm. Meth. 206: 163-169; U.S. Pat. No. 6,080,840). However, although such TCRs can be recognized by TCR-specific antibodies, they can only recognize a native ligand at a relatively high concentration, suggesting that the recognition is instable.
Furthermore, for production of TCRs with original antigen specificity, there are many investigations on how to improve stability of water soluble TCR fragments, including variable domains of a single-chain TCR (Novotny, et al (1991) PNAS USA 88:8646-8650), extracellular domains in a heterodimeric TCR (Garcial et al (1996) Science 274:209-219), or modification of such molecules (Shusta et al (2000) Nature Biotechnology 18:754-759), Boulter et al (2003) Protein Engineering 16:707-711). In these researches, Novotny et al used a flexible peptide for linking variable domains to construct a single-chain TCR. However, stable molecules could be obtained only after replacing hydrophobic residues exposed on surface with hydrophilic residues containing water soluble side chains. Shusta et al modified the single-chain TCR variable domain structure by introducing random mutations into the whole molecule and by displaying on yeast surface and selection with FACS. Garcia et al constructed extracellular domain 2C of an α/β TCR and native inter-chain disulfide bonds were kept in the structure. Boulter et al improved α/β heterodimer construct by introducing an artificial disulfide bond buried between two constant domains.
The approach of using disulfide between constant domains has been used for phage display of TCR vectors which have been used for generation of many high affinity TCRs (Li et al (2005) Nature Biotechnology 34:349-354; Liddy et al (2012) Nature Medicine 18:980-987). However, the inventors have found that the probability for successful production of a high-affinity TCR using such constructs is still very low, and it is difficult to obtain TCR with both high affinity and high stability. So it is necessary to develop new strategies for producing a TCR and fragments thereof having water solubility, high affinity and high stability